Ubiquitin (Ub) plays a central role in cellular biology. Ubiquitination of proteins is balanced by Ub removal, a process mediated by the family of deubiquitinating enzymes (DUBs). These DUBs are likely to be involved in myriad regulatory functions. The apparent redundancy of these proteases is likely due to unique functions and specificities for individual DUBs. This proposal aims to resolve the sequence selectivity of functional human DUBs using peptide arrays linked to monomeric or polymeric Ub. We hypothesize that the action of Ub ligases and deubiquitinating enzymes are conceptually similar to that of protein kinases and protein phosphatases but this hypothesis necessitates a better understanding of the substrate specificity of deubiquitinating enzymes. This same approach should be equally applicable to the study of the growing number of Ub-like proteins. Modification of proteins by Ub and Ub-like proteins is already known to be involved in numerous human disease states, including some cancers and neurodegenerative disorders. Knowledge of DUB activity and preferred substrates may help to uncover the molecular details of some of these diseases and hopefully lead to viable treatment strategies.